TALs pre-exposed to autologous TLRMs activated with LPS or LPS +IL-10R Ab exhibited a fourfold higher expansion after coculture relative to TALs not exposed to ascites CD14+ cells or exposed to untreated ascites CD14+ cells (figure 5E). and chemokine array analysis, ex vivo cocultures with T cells, in vivo tumor challenge and T cell transfer experiments, RNA-sequencing and mass spectrometry. Results We demonstrated the feasibility of isolating ascites monocytes and restoring their ability to function as bona fide antigen-presenting cells (APCs) with Toll-like receptor (TLR) 4 lipopolysaccharide and TLR9 CpG-oligonucleotides, and a blocking antibody to interleukin-10 receptor (IL-10R Ab) in the ID8 model. The ascites monocytes were laden with tumor antigens at a steady state in vivo. After a short 48?hours activation, they upregulated maturation markers (CD80, CD86 and MHC class I) and demonstrated strong MK8722 ex vivo T cell stimulatory potential and effectively suppressed tumor and malignant ascites in vivo. They also induced protective long-term T cell memory responses. To evaluate the translational potential of this approach, we isolated ascites monocytes from stage III/IV chemotherapy-na?ve OC patients. Similarly, the human ascites monocytes presented tumor-associated antigens (TAAs), including MUC1, ERBB2, mesothelin, MAGE, PRAME, GPC3, PMEL and TP53 at a steady state. After a 48-hour treatment with TLR4 and IL-10R MK8722 Ab, they efficiently stimulated oligoclonal tumor-associated lymphocytes (TALs) with strong reactivity against TAAs. Importantly, the activated ascites monocytes retained their ability to activate TALs in the presence of ascitic fluid. Conclusions Ascites monocytes are naturally loaded with tumor antigen and can perform as potent APCs following short ex vivo activation. This novel ascites APC vaccine can be rapidly prepared in 48? hours with a straightforward and affordable manufacturing process, and would be an attractive therapeutic vaccine for OC. paracentesis from four MK8722 chemotherapy-na?ve patients with stage III/IV epithelial OC prior to primary debulking surgery (figure 4A). Ascites cellular content was heterogeneous among patients; tumor cells Rabbit Polyclonal to BLNK (phospho-Tyr84) (CD45-Epcam+) and myeloid cell populations accounted for 1%C85%?and 10%C30% of the total cells isolated from OC ascites, respectively (figure 4B). CD14+ peritoneal monocytes coexpressed CD11b and CD11c, with a fraction also expressing CD16, CD303 or CD141 (figure 4C). Consistent with our results in the ID8 ovarian model, CD14+ cells from human ascites lacked costimulatory CD80 and CD83 expression and were low in CD86 and CD40 expression (figure 4D). Ascites monocytes from OC patients exhibited transcriptome profiles of protumorigenic macrophages,29 30 which in principal component analyzes were closer to cancer macrophages than to normal circulating monocytes or normal tissue macrophages31 (figure 4E). Open in a separate window Figure 4 CD14+ cells retrieved from human OC ascites express low levels of costimulatory and antigen-presentation markers and present TAAs in situ. (A) Malignant ascites were collected via paracentesis from chemotherapy-na?ve patients with stage III or IV epithelial OC prior to primary debulking surgery. (B) Flow cytometry characterization revealed different percentages of immune cells representing CD14+ cells, T, B, natural killer and tumor cells in the malignant ascites of four OC patients samples. (C) Representative dotplots of ascites-derived CD14+ cells showing the expression of myeloid-lineage markers and D) costimulatory markers. (E) Principal component analysis showing clustering of ascites CD14+ cells with endometrial cancer macrophages. (F) Left panel in blue and white, immunopeptidomic analysis with mass spectrophotometry highlighting the MHC Class I and II presentation of TAAs on CD14+ cells and tumor cells from two OC patients. The data was presented as relative MS signal (log2). Right panel with red and light pink, comparing the transcriptional levels of expression of TAAs in CD14+ cells and tumor cells from the same two OC patients. The data were presented as relative expression (transcripts per million (TPM)/TPM average). OC, ovarian cancer; TAAs, tumor-associated antigens. To determine if human ascites monocytes are loaded and present tumor antigens in situ, we purified CD14+ cells from ascites by FACS sorting (purity ~99%; online supplementary figure 3A and B) and analyzed the immunopeptides bound to surface HLA molecules by MS/MS profiling of peptides eluted from class I and II HLA molecules. We identified in two out of three patients several TAA candidates presented on HLA class I and II molecules of ascites CD14+ cells, which were also presented by autologous tumor cells isolated from the same ascites. These TAAs included cancer-testis antigens (CT55, MAGEs, PRAME, SAGE1); known OC-associated antigens (MUC1, ERBB2, mesothelin, TP53); and other TAAs (GPC3, PMEL, TYRP1, ODF2) (figure 4F and online supplementary file 1). We demonstrated by RNAseq analysis the selective expression of some of the presented TAAs in tumor.